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Microbiome of smear ripened cheese and its changes in dependence on the stages of production
Jandová, Karolína ; Mikešová,, Martina (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part describes soft smeared cheeses, then deals with the technology of manufacturing said cheese with importance on the development of microbiome and describes the most commonly wanted microorganisms and contaminants. Shortly also deals with legislation requirements on the microbial quality of these chesses. The experimental part studies the development of microbiome and contamination by fungi of soft smeared cheese. Firstly, samples were collected in manufacture from each step of manufacturing. In the first part, microbial and microscopical assessment was done. In the second part, DNA was isolated from samples by a commercial kit and phenol-chloroform extraction. The concentration and purity of isolates were determined by spectrophotometry. By qPCR of the DNA isolates the main microbiome and contamination by fungi were examined. As part of the microbial and microscopical assessment, the expected bacteria were determined, and several species of yeast were found, instead of the expected one. Mold contamination was also found. Using molecular diagnostic techniques, the presence of bacteria, yeasts, fungi and the genera Brevibacterium and Candida was proved.
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Testing of primers for real-time PCR-HRM analysis of fruit products containing one fruit species
Boháčová, Barbora ; Dzurendová, Simona (referee) ; Fialová, Lenka (advisor)
Determining the authenticity of fruit products, which are often counterfeited by substituting part of a more expensive fruit with a cheaper but botanically similar fruit, is a current topic in the food industry. A prime example is the dilution of apricot products with peach puree. This study focuses on testing specific primers AGS18 and PdCass to reveal the true proportion of apricot content in model products mixed with peach puree using PCR analysis followed by HRM analysis. Testing of these primers for authenticity determination revealed limited utility of AGS18 primers due to the formation of small amount or no specific products during reactions with fruit DNA. PdCass primers required PCR condition optimization, but subsequently, specific DNA sequences from fruit leaves could be amplified in sufficient quantities. PCR analysis of DNA from model products with PdCass primers provided specific products in samples with apricot content of 70% or less. HRM analysis of samples and calculation of GCP did not distinguish purees with 0%, 10%, and 30% apricot content. However, purees with 50% and 70% apricot content exhibited significantly different melting curves compared to other samples.
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Selective isolation of the genus Bifidobacterium bacteria from foods
Mizerovská, Lucie ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria (LAB) are very often used in food procesing industry, such as milk products, cheese and fermentsd salami production in nova days. In diploma thesis were tested symbiotic food supplements from different producers. Bacterial DNA was isolated from crude cell lysates of six food suplements by magnetic particles P(HEMA-co-GMA). PCR-ready DNAs were isolated. from all products The detection of Bifidobacterium bacteria identified by PCR was in agreement with those declared by the manufacturers. Magnetic particles with immobilized antibodies against Bifidobacterium were used in the next part of thesis. These particles were used for the isolation of target cells from two products with cell identification by genus specific PCR.
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DNA microextraction from plant vegetable matrix
Cesnak, Filip ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.
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Selective isolation of of the genus Lactobacillus bacteria from foods
Novotná, Eva ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria of genus Lactobacillus play an important role in the digestive tract of human. They are used in food processing and they are the part of food supplements. Lactic acid bacteria of the genus Lactobacillus can be identificated by polymerase chain reaction (PCR). Bacterial DNA was isolated from cell lysates of 4 synbiotic food suplements by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. Magnetic particles with immobilized antibodies against Lactobacillus bacteria were used in the next part of thesis. These particles were used for isolation target cells from products with their identification by genus specific PCR.
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Identification of bacteria of Lactobacillus acidophilus species in probiotic products
Sznapková, Veronika ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria (LAB) are an important part of fermented dairy products, pharmaceuticals and food supplements. At present, rapid and accurate identification of bacteria is carried out using molecular biological methods based on DNA amplification. The aim of the thesis was to identify by non-cultivation bacteria of genus Lactobacillus and bacteria of species Lactobacillus acidophilus in complex matrices at total of seven different food supplements. Total DNA was isolated from crude cell lysates using magnetic carrier P(HEMA-co-GMA). Amplificability of DNA was verified by PCR using primers specific for the domain Bacteria. In next step isolated DNA was amplified using primers specific for the genus Lactobacillus and species Lactobacillus acidophilus to demonstrate the presence of this bacterial genus and species declared by the producers. The results of bacteria identification obtained by PCR were compared with declared specification given by the producers.
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Influence of matrix type on the authentication of foodstuffs containing fruits
Kopková, Pavlína ; Strečanská, Paulína (referee) ; Fialová, Lenka (advisor)
Certain types of food, mainly the more expensive ones, are often adulterated to reduce their manufacturing price. However, this reduces their quality and can also have a negative impact on the health of the consumer. Children's fruit products are also targeted by fraudulent producers, where the declared fruit is most often replaced by a cheaper version. This work focuses on the detection of adulterated foods using various analytical methods, in particular PCR. The theoretical part focuses on the issue of food adulteration, the analytical methods used for detecting adulteration, and also on mango and banana which are determined in this work. The aim of this thesis was to determine what effect the type of matrix has on the determination of fruit components in food by PCR. Three types of matrix were used for this purpose - fruit puree, smoothie, and bars. An important task was to optimize the DNA isolation to achieve adequate purity and concentration of DNA. Then, the amplifiability of the obtained DNA was verified. The DNA isolates were then analyzed by multiplex PCR with primers specific for mango and banana. The results were verified by agarose gel electrophoresis. Subsequently, it was possible to determine that the fruit component in bars and fresh smoothies was the most easily analyzed by PCR and, on the contrary, the determination was problematic for puree. The instrumental part was focused on the determination of phenolic compounds in the products by HPLC. For this purpose, optimization of the extraction of phenolic compounds was necessary. This method was able to detect the presence of mangoes in all samples.
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Probiotics and their use in food industry
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic bacteria are defined as live microorganisms, which when consumed in the determining quantities, have healthy and beneficial effects. Most of probiotics belongs to the genera Lactobacillus and Bifidobacterium. These and other genera of microorganisms are successfully used in industry, including food industry at present. Probiotics are used primarily in dairy products and food additives in food idustry. Probiotic bacteria, like other organisms, can be to identifie by PCR method that allows amplifying specific regions of DNA. Polymerase chain reaction was performed after DNA isolation from bacterial cultures of three strains using phenol extraction method. PCR specific for the domain Bacteria and genus-specific PCR were used for the confirmation of the presence of bacteria of the genus Lactobacillus.
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PROBIOTIC GENES OF SIGNIFICANT LACTIC ACID BACTERIA IN FOOD
Konečná, Jana ; Ševčovičová,, Andrea (referee) ; Doškař, Jiří (referee) ; Španová, Alena (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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